Tacrolimus is a macrolide isolated from Streptomyces tsukubaensis. Tacrolimus has the chemical name [3S-[3R*[E(1S*,3S*,4S*)],4S*,5R*,8S*,9E,12R*,14R*,15S*,16R*,18S*,19S*,26aR*]]-5,6,8,11,12,13,14,15,16,17,18,19,24,25,26,26a-hexadecahydro-5,19-dihydroxy-3-[2-(4-hydroxy-3-methoxycylohexyl)-1-methylethenyl]-14,16-dimethoxy-4,10,12,18-tetramethyl-8-(2-propenyl)-15,19-epoxy-3H-pyrido[2,1-c][1,4]-oxaazacyclotricosine-1,7,20,21(4H,23H)tetrone. The structure of tacrolimus, giving the numbering, is shown below in FIG. 16.
Tacrolimus is also known as FR-900506 or FK-506. Tacrolimus has immunosuppressive activity and antimicrobial activity.
The immunosuppressive activity of tacrolimus is particularly important and has led to the increasingly wide use of this drug. Immunosuppression is used clinically in a number of contexts, most importantly in preventing rejection in organ transplantation. Immunosuppressive drugs are also administered in prevention of Rh hemolytic disease of the newborn and in the treatment of autoimmune disorders. Tacrolimus inhibits T-cell activation by binding to a cytosolic protein known as FKBP (FK506 binding protein). The drug-binding protein complex stably associates with calcineurin. This inhibits the serine-threonine phosphatase activity of this Ca2+-dependent enzyme. This inhibits calcineurin-dependent activation of lymphokine expression, apopotosis, and degranulation (G. Wiederrecht et al., “The Mechanism of Action of FK-506 and Cyclosporin A,” Ann. N.Y. Acad. Sci. 696:9-19 (1993)).
Tacrolimus can be administered intravenously in a short or continuous infusion or orally. Tacrolimus, like other immunosuppressant agents, has a spectrum of toxicity. The major toxicity associated with clinical use of the drug is nephrotoxicity. In addition, neurotoxicity can develop, associated with headache, tremor, insomnia, pain, or other symptoms. Additionally, gastrointestinal toxicity manifested by diarrhea or nausea can develop, as can cardiovascular toxicity manifested by hypertension.
Additionally, metabolic toxicity can develop as manifested by the development of such symptoms as hyperkalemia, hypomagnesemia, or hyperglycemia. In addition, long-term immunosuppression with tacrolimus can produce increased risk of all types of infections, not only the usual bacterial, viral, and fungal pathogens, but also various unusual opportunistic infections as well.
Additionally, there is an increased risk of lymphomas and related malignancies associated with the administration of tacrolimus (M. L. Cleary & J. Sklar, “Lymphoproliferative Disorders in Cardiac Transplant Recipients are Multiclonal Lymphomas,” Lancet 2:49-493 (1984); L. J. Swinnen et al., “Increased Incidence of Lymphoproliferative Disorder After Immunosuppression with a Monoclonal Antibody OKT3 in Cardiac-Transplant Recipients,” N. Engl. J. Med. 323:1723-1728 (1990)). At least some of these malignancies are related to impaired immune responses to Epstein-Barr virus (B. Z. Katz et al., “Latent and Replicating Forms of Epstein-Bair virus DNA and Lymphomas in Lymphoproliferative Diseases,” J. Infect. Dis., 160:589-598 (1989)).
The potency and the spectrum of toxicities of tacrolimus requires sensitive, reproducible, and reliable methods for monitoring the blood concentration of these compounds after administration to a patient, such a patient undergoing organ transplantation. It is important that such methods be sensitive enough to detect low concentrations of tacrolimus. It is also important that such methods be reliable and reproducible, and avoid interference from compounds such as metabolites of tacrolimus.
Although antibodies and immunoassays to tacrolimus exist, and are described, for example, in U.S. Pat. No. 5,532,137 to Niwa et al., incorporated herein by this reference, there is still a need for the development of improved antibodies and immunoassays specific for tacrolimus. There is, particularly, a need for improved monoclonal antibodies to tacrolimus that can be used in developing a sensitive, reliable, and reproducible immunoassay for tacrolimus.
The development of a reliable immunoassay for tacrolimus is complicated by the fact that tacrolimus has a number of metabolites that are found in the blood of an individual being treated with tacrolimus. The conversion of tacrolimus to these metabolites involve demethylation, hydroxylation, and ring formation. It is important that antibodies to tacrolimus have as little cross-reactivity with these derivatives as possible.
There is therefore a need for the development of improved monoclonal antibodies to tacrolimus that are useful for immunoassays for tacrolimus and that possess a minimal degree of cross-reactivity to tacrolimus metabolites. There is also a need for improved immunoassays using such monoclonal antibodies for the detection and determination of tacrolimus in the blood of patients being administered tacrolimus.